Driving immobilized lipases as biocatalysts: 10 years state of the art

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Review

Driving immobilized lipases as biocatalysts: 10 years state of the art and future prospects Bruno R Facin, Marina Melchiors, Alexsandra Valerio, José Vladimir Oliveira, and Débora de Oliveira Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b00448 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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Industrial & Engineering Chemistry Research

Driving immobilized lipases as biocatalysts: 10 years state of the art and future prospects

Bruno R. Facin, Marina S. Melchiors, Alexsandra Valério, J. Vladimir Oliveira, Débora de Oliveira*

Department of Chemical and Food Engineering, UFSC, P.O. Box 476, 88040-900, Florianópolis, SC, Brazil.

ACS Paragon Plus Environment

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Abstract Immobilized lipase is a cost-effective biocatalyst for a wide variety of industrial applications, mainly in food, cosmetics, and pharmaceutics due to the huge number of the so-called chemicallycatalyzed reactions of industrial interest that can be replaced by immobilized-enzyme catalysis. In this scenario, there is a growing demand to develop new products, supports, and immobilization protocols, as well as to elucidate the reaction mechanism aiming to cross the barrier imposed by the cost of immobilized lipases. As a large number of researchers has focused their efforts in order to find out new applications taking into account new products emerging continuously, it is important to clarify the traditional and alternative processes routes using immobilized lipases. At the last decade, almost five thousand researches were reported and this number continuous rising. This review reports the last ten years research works on the techniques used for lipase immobilization proposing a brief introduction about traditional immobilization techniques and an overview of the reactions catalyzed by immobilized lipases with focus on applications and some products obtained by each pathway.

Keywords: lipase; carrier; immobilization technique


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1. Introduction Lipases (triacylglycerol hydrolases, EC 3.1.1.3) constitute a group of enzymes that catalyze the hydrolysis of lipids in biological systems. Lipases are hydrolases whose natural function is to hydrolyze ester bonds in triglycerides. 1–5 These enzymes are among the most commonly used for biocatalysis purposes due to their wide specificity coupled in many instances to a high regio and enantioselectivity and specificity, high stability in different reaction conditions and broad spectrum of reactions that they are able to catalyze. These properties have been relevant for industrial applications of lipases, for example, in food technology, biodiesel production, and fine chemistry. 6–13

Lipases catalytic mechanism involves a conformational equilibrium, which makes it quite peculiar.

14,15

In the closed form, a polypeptide lid blocks the active center, making the enzyme

even inactive. However, in the open form, this lid is shifted and exposes to the medium a large hydrophobic pocket containing the active center. This way, this hydrophobic region may be adsorbed and stabilized to any hydrophobic surface, such as oils (named interfacial activation), hydrophobic proteins, and another open form of a lipase or even hydrophobic supports. 16–18 Nowadays, lipase properties can be improved by using different tools. Microbiology, by means metagenomics, has made a huge amount of new enzymes available, some of them of unknown origin, but with new and interesting features. The genetic tools, such as mutagenesis and directed evolution, have improved the enzyme properties for specific problems (stability or activity in a particular medium, activity versus a substrate). Thus, old techniques, such as chemical modification or immobilization, have apparently lost relevance in the preparation of an industrial biocatalyst.19 Thus, even though these enzymes may be used in free form, the possibility of lipases immobilization emerges as an alternative to overcome some misfortunes in the lipase-catalyzed


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reaction, such as limited solubility, thermal, mechanical, and operational stability of the enzyme, as well as impossibility of catalyst reuse, leading to high production costs hindering large-scale applications.20–22 Immobilized enzymes on supports, especially in low-cost supports can offer some advantages such as increase activity, specificity, and selectivity, improve structural stability and ease recovery.23–25 The support surface chemistry could also be designed with specific groups, which could effectively adsorb besides enzyme the substrate increasing enzyme activity. It is the case of hydrophobic groups, which may favor the physical adsorption phenomenon. 22,26 A large number of researchers focused their efforts in order to develop new immobilization protocols (which may lead to a lower degree of inactivation improving the lipase activity),27 new materials for support (blends and composites) or new morphologies for those already consecrated (especially nanoscale),28,29 besides new applications for these versatile biocatalysts.30 According to Gonçalves et al.,31 the terms immobilization and lipase are among the most frequently (1st and 4th places) used author-keywords to describe enzyme immobilization studies in the last 10 years. For this period, combining and using these same terms as the topic of search, almost five thousand research papers were reported by Web of Science and this number is continuously rising. Data were downloaded on July 2, 2018. This review article presents an overview about the current state of the art regarding immobilized lipases as biocatalyst, providing knowledge about the latest products obtained by each reaction catalyzed by this biocatalysts, besides the reaction mechanism involved. This work also reviews the advances in traditional methods for enzyme immobilization aiming to improve the lipase activity and stability. Furthermore, this review presents a brief discussion about how to pick the suitable support for lipase immobilization, as well as the latest advances in materials to this.


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Finally, some perspectives concerning the employ of this biocatalyst in reactions of interest, future trends, challenges and prospects in this field are also presented.

2. Lipase immobilization methods and supports 2.1. Choice of support Supports or carriers to lipase immobilization can be obtained from different materials coming from many sources. Selected materials should be able to protect the enzyme structure against adverse reactions conditions keeping the structural integrity and catalytic activity; for example, hydrophobic carriers on lipase immobilization can raise the enzymatic activity.29,32 In this sense, the choice of the support matrix, with the ideal characteristics to the desired purpose, is crucial for the immobilization process success.21,22,33,34 Enzyme supports can be classified according to the chemical composition in organic or inorganic materials, and the source can be natural or synthetic.28 Inorganic supports, as silica,35,36 bentonite,37 montmorillonite,38 sepiolite,39 hydroxyapatite,40 and activated carbons41 exhibit good thermal and chemical stability and great mechanical resistance. These materials have also good sorption properties due to the well-developed porous structure which ensure high superficial area and multiple sites for enzymatic immobilization.29 Organic supports, on the other hand, especially polymers, are able to facilitate covalent binding without crosslinking agents due to a large number of functional groups.42 Besides that, these kinds of supports show high protein affinity, reducing possible negative effects of lipase immobilization process. Among synthetic polymers it is important to highlight poly(vinyl alcohol) (PVA),43 polystyrene (PS),44 poly(methyl methacrylate) (PMMA),45 and polyurethane (PU);46,47 whereas for biopolymers it is possible to cite collagen,48 cellulose,49 carrageenan,50 chitosan,51 and alginate.52


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A question during the support material selection is the process in which the biocatalyst will be used to focus on the reaction medium and operational conditions.53 PMMA, a synthetic polymer, for example, has poor tolerance against some organic solvents and dissolves easily in such media.54 But, as important as that, the mechanical properties of the support for immobilization have similar demands. The selection of the mechanical properties will depend more on the final configuration of the reactor than the application for immobilization. So, the idea about a universal appropriate support to lipase immobilization should not be considered since there are several parameters that may define a support: internal geometry, specific surface area, superficial activation degree, and pore diameter.21,22 However, some properties could be considered in the search of an ideal support, for example: hydrophilicity, inertness towards enzymes, biocompatibility, low cost, resistance to microbial attack, and high mechanical and chemical stability.24,25,28 Moreover, others important characteristics should be the high protein affinity, reactive functional group availability, not toxicity, and reuse viability.55 All these materials, independently of their source, are still extensively investigated, due to the abundance in nature, as biopolymers, for example, or due to the easy synthesis, as the inorganic oxides and synthetic polymers. However, particularly in the last decade, the attention has been focused on new supports morphologies,56,57 especially at nanoscale,58–62 hydride,63,64 and composite supports23,65 combining materials properties in order to improve the support characteristics such as high specific surface area and excellent mechanical properties. Moreover, a recent cost analysis regarding, suggests that the biggest cost involved in the commercial adoption of this biocatalyst is the cost of the support material, and not the enzyme.66 Chiaradia et al.67 immobilized Lipase B from Candida antarctica on poly(urea-urethane) nanoparticles incorporated with iron oxide aiming to enhance the separation from the reaction medium by a magnetic field.


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Similar support (superparamagnetic nanoparticles of Fe3O4) was used by Galvão et al.68 for Pseudomonas fluorescens immobilization and further application to the kinetic resolution of secondary alcohols as rac-indanol, rac-1-phenylethanol, rac-1-(3-bromophenyl)-1- ethanol, and rac-1-(3-methylphenyl)-1-ethanol. This was the first time that this nanohybrid catalyst was used to the kinetic resolution of secondary alcohols. Sóti et al.69 used poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA) nanofibers to successful immobilize Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CalB) and then obtain a biocatalytic complex with excellent stability even after ten reuse cycles of acylation and hydrolysis reactions. Hrydziuszko et al.70 studied 5 carriers of different material, porosity, and functionality for Burkholderia cepacia lipase immobilization and they achieved yields of protein binding varied from 9 to 85%. Recently, the immobilization of lipases on heterofunctional supports has been revealed as a powerful tool to achieve one step immobilization, purification, hyperactivation and strong stabilization of the lipases.

71–73

It allows an increment in activity after immobilization since the

open form of the lipases may be stabilized on these supports.74 Albuquerque et al.71 investigated the immobilization of the lipases from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosus on octyl supports activated with divinyl sulfone (OCDVS). These three lipases have some problems using octyl-glyoxyl (enzyme inactivation or moderate covalent immobilization yield) and the work revealed that the new heterofunctional OCDVS support was very suitable to solve these problems. Once that the appropriate support has been chosen, there are many forms to immobilize lipases on or into it and each one shows a specific interaction mechanism support-enzyme which may affect deeply the lipase behavior: increasing, decreasing or even taken your complete inactivation. Some materials and source of lipases that had been immobilized using these supports are


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summarized in Table 1 together with information about support morphology, immobilization type and immobilization efficiency.

2.2. Techniques for enzyme immobilization The main goal of immobilization process is to obtain a stable biocatalyst that can be reused for several times with minimal loss of initial activity. Thus, the immobilization process should be able to keep the original enzyme stability or allow the enzyme becomes highly stabilized during long periods of time.58,75 Usually, the immobilization methods are divided into two major classes taking into account the enzyme interaction with support: chemical and physical methods.15 Physical methods show normally physical confinement of enzyme within the support or weak and noncovalent interactions between enzyme and support such as hydrogen bonds, hydrophobic interactions, van der Waals forces, and ionic binding. On the other hand, chemical methods involve the formation of covalent bonds between enzyme and support such as ether, amide or carbamate bonds (see Fig. 1).21,22 For the chosen immobilization method, some factors have been considered, as: lipase loading, relative enzymatic activity, cost of immobilization procedure, enzyme deactivation, the toxicity of immobilization reagents, and the final characteristics of the catalyst.21,55 Once the physicochemical properties of both support surface and enzyme are known it can be used as an advantage to obtaining systems that outperform the free enzyme.27,55 To design the support surface with a specific group which could effectively adsorb substrate might be an advisable method to increase enzyme activity.76 Thus, in the literature, there are different techniques for lipase immobilization. In this review, it will be explored physical adsorption, covalent bonding, cross-linked, and entrapment technique. Table 2 presents some reports of enzyme immobilization techniques,


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highlighting the immobilization efficiency, the activity retained, lipase source and support employed.

2.2.1. Physical adsorption Physical adsorption is probably the simplest enzyme immobilization method and involves adsorbing physically enzymes or attaching onto the support material. This technique requires the contact between the support and an enzyme solution and, since the interaction enzyme-support is strong contact time dependent, the adsorption occurs by incubating15 or by drying the enzyme solution on the support material.15,21 In order to improve the adsorption capacity of lipases on these supports, certain parameters (pH, ionic strength, temperature, initial protein loading and contact time), isotherm, kinetic, mechanism, and thermodynamic studies must elucidate the adsorption process.77–79 The enzyme adsorption phenomenon implicates in weak non-specific forces such as van der Waals, hydrophobic interactions, hydrogen bonds, and ionic bonds.21 In the case of lipases, hydrophobic interaction is most common because lipases are adsorbed spontaneously from aqueous solutions onto hydrophobic surfaces quicker than most other proteins.15,34 Surfaces containing hydrophobic groups enable lipase to take more side-on orientations with larger spreading due to the strong hydrophobic interaction, while on surfaces containing hydrophilic groups this interaction is weakened and lipase could take more end-on orientations with smaller spreading.22,26,76,80 Some research works15,55,75,81 suggested the lipase catalytic activity increase after physical adsorption, and this event may be related to the hydrophobic interaction supportlipase leading the opening of the polypeptide chain called lid what exposes the active site and


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changing the lipase to its active form similar to interfacial activation motivated by hydrophobic substrate phase, as illustrated in Fig. 2. The major inconvenience of this technique is the reversibility of the immobilized enzymes by the fragile interaction with the support, being easily removed from support under mild conditions, especially in aqueous media.21,34,55 Generally, this is not a desirable characteristic, particularly when used in analytical assays and sensor devices.58 However, it should be an attractive method when the cost of the support is significant, whereas as soon the enzymatic activity reduces due to the enzyme leaching, the support can be regenerated and reloaded with fresh enzyme.21,82 The hydrophobic adsorption is suppressed by the presence of non-ionic detergents or organic solvents. In this case, the enzyme should remain adsorbed on the support via ionic exchange, and very likely, the lipase open form should be maintained by steric reasons. However, the support may be re-used after enzyme inactivation by washing the biocatalyst with ionic detergents, keeping the advantage of a reversible immobilization.83–85 Rueda et al.85 immobilized 5 different lipases, those from Candida antarctica (A (CalA) and B (CalB)), from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and a phospholipase (Lecitase ultra, LU) on octyl-glutamic heterofunctional agarose beads by physical adsorption and they verified the desorption phenomenon by incubation in ionic detergents. The authors confirm the supports could be reused in the immobilization of new batches of fresh enzyme without any difference in stability or activity compared to the original on the cycles evaluated (5 times). Related to the forces in the adsorption processes, when lipases are immobilized on hydrophobic supports, according to Hanefeld et al.55 van der Waals forces are responsible by the interaction support-enzyme leading to a gain in the entropy. Conversely, when hydrophilic carriers are used, hydrogen bonds occur exclusively due to a large number of NH-groups of the polypeptidic chain


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which can readily interact with carbonyl groups of the polymers, for example.55,58 The commercial immobilized lipase, Novozym® 435, is obtained by the adsorption method.86 This biocatalyst consists in Candida antarctica lipase B (CalB) adsorbed on hydrophobic macroporous polymer based on methyl and butyl methacrylic esters cross-linked with divinylbenzene to avoid or minimize the leaching effects.87

2.2.2. Covalent bonding Covalent bonding is a usual technique of irreversible enzyme immobilization and this method consists in the formation of covalent bonds between enzyme and support material.88 These interactions involve side chain amino acids, such as lysine, cysteine, aspartic and glutamic acids, and several functional groups, like carboxyl group, amino group, epoxy group, indole group, phenolic group, sulfhydryl group, thiol group, imidazole group, and hydroxyl group, which are not essential for the catalytic activity of enzyme.21,28 Comparing with immobilization by adsorption, covalent immobilization exhibit in general some superiority since the enzyme can remain on supports under strict conditions and even be used in any reaction media.42,58 Covalent bonding makes a robust support-enzyme link, which may ensure the enzyme is tightly fixed preventing enzyme release (protein contamination of product) into the reaction media and, consequently, affording more reuse cycles. Covalent bonding makes possible to submit the immobilized lipase to unfolding/refolding reactivation strategies without risk of lipase desorption, making it unnecessary to discard neither lipase nor support.89 Rueda et al.

89

Investigated the

unfolding/refolding behavior by incubation in high concentrations of organic solvents of CalB and TLL lipases immobilized on octyl–glyoxyl agarose beads and they realized the recovery of


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enzymatic activity decreased with further reactivation cycles suggesting that the exact same structure of the enzyme was not obtained during each refolding cycle. In general, covalently immobilized enzymes should be used in any reaction media, especially those aqueous, and when denaturing factors are present since the covalent bonds are strong enough to keep the enzyme linked decreasing the conformational flexibility and thermal vibrations.21 Nevertheless, the major challenge around this technique is the chemical modification that the enzyme is submitted.55 Covalent bonding often leads to some degree of enzymatic inactivation and if critical functional groups to catalysis are modified this degree can become severe.90 In order to prevent this inactivation, especially at the active site region, a strategy is adopted which consists in carrying out the immobilization process in the presence of substrates or competitive inhibitors.91 It could be a valuable advantage when lipases are the targets of immobilization once they might be immobilized under their closed form protecting the active site and after process ending changed to open form.15 Some immobilization protocols report the employ of spacers between enzyme and support to improve the performance of the process. When long spacers are employed, higher conformational flexibility is expected for the enzyme. Lipases fit very well in this case for having this peculiar behavior (change their structural conformation) when interacting with the substrate. In contrast, short spacers do not allow too much mobility but can ensure more thermal stability and low risk of enzymes leaching under any distorting agent (heat, organic solvents, extreme pH values).75 Multipoint covalent attachment is commonly observed when short spacers are employed.22,75,92 Some research works report assistance of glutaraldehyde as a coupling agent due to its


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extraordinary capacity to build crosslinking,93 but this mechanism will be more explored on next topic. In the last ten years, support activated with divinylsulfone (DVS) has been used to immobilize several lipases. The tuning of the catalytic properties of lipases immobilized on DVS-activated support shown as a new possibility for immobilize lipases via covalent bond. This support can react with different moieties of the proteins, and produce some rigidity, and as a final enzyme– support reaction end point, the blocking of the support may be performed with different amino or thiol compounds.

71,94–96

Santos et al.95 achieved high values of activity recovery and

immobilization yield (100% yield) for TLL lipase immobilized on DVS-activated agarose beads. Additionally, it was realized that further incubation under alkaline conditions (pH 10) also produced changes in enzyme features even permitting to improve the enzyme stability. In the same way as for physical adsorption, both hydrophilic and hydrophobic supports can be used for covalent immobilization.97 Jose et al.97 reported the use of functionalized chitosan, epoxy acrylic resin, and functionalized magnetite as supports for Candida antarctica lipase B covalent immobilization with subsequent application in the esterification of R/S-ibuprofen with ethanol. Another technique of immobilization was employed as well as others supports, but some decrease in the specific activity was observed in those immobilized by covalent bonding. Miao et al.98 used superparamagnetic Fe3O4 nanoparticles to covalently immobilize lipase B from Candida antarctica. The biocatalyst obtained when compared to the free lipase was more stable, showed higher activity at several temperature and pH and also allowed 5 reuse cycles.


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2.2.3. Crosslinking Crosslinking is another technique of irreversible enzyme immobilization, which uses a bifunctional agent to make the required bonds. It is quite common to see this technique being used in combination with others, such as adsorption, to avoid enzyme leaching of carrier.15 However, the main goal here is to form carrier-free enzyme complex in order to eliminate most of the drawbacks associated with the employ of physical adsorption technique, as already discussed previously. Glutaraldehyde is the most usual and popular crosslinking agent as it is economical and readily available in large quantities. However, others are also known, such as dextran polyaldehyde and divinyl sulfone, and can be used when glutaraldehyde shows unsatisfactory results.21,88,99 As in the case of covalent bonding to supports, the amino groups of the enzyme are strictly involved in the bond formation. The primary amino groups of lysine residues on the enzyme surface react with dialdehydes groups of crosslinking agent leading reversible Schiff’s base formation. The subsequent reduction is required to turn this crosslinking irreversible. This second step is not necessary when the crosslinking agent employed is glutaraldehyde since hydration, oligomerization and aldol condensations reactions occur and simple Schiff’s base is not the dominating mechanism.15 However, the exact mechanism of glutaraldehyde action still presents some controversies in the scientific community. The most accepted involves Michael-type addition of –NH2 to α,β-double bonds, yielding a stable secondary amine (see Fig. 3).93 Crosslinking technique has been studied since the 1960s, but due to several limitations, such poor reproducibility, low mechanical stability and low residual activity, it was left out and the focus switched to carrier-bound enzymes techniques. Only in the 2000s, this research took the next step and now it attracts growing attention. To date, carrier- free immobilization mainly includes a


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crosslinked enzyme (CLE), crosslinked enzyme crystals (CLECs), crosslinked spray-dried enzyme (CSDE) and crosslinked enzyme aggregates (CLEAs). The difference among them is basically the method of production, such as direct crosslinking, crystallization, spray-drying, and aggregation, respectively.27 Sheldon and Pelt99 approached more deeply this topic in their review with focus on the application of this technology to a variety of enzymes, including lipases. Cui and Jia27 also published a review on this subject and discussed strategies and optimization processes to this technique. Anand and Weatherley100 investigated the hydrolysis of sunflower oil catalyzed by immobilized lipase from the fungal yeast Candida rugosa OF360 in three different polyolefin supports. The authors carried out some reuse cycles and better enzymatic stability was found to the biocatalysts for which glutaraldehyde was used as a crosslinking agent in order to improve adsorption obtained results. Mbanjwa et al.101 produced Pseudomonas fluorescens lipase microspheres carrier-free using the enzymes in a water-in-oil emulsion and an oily suspension of glutaraldehyde and ethylene diamine as crosslinking fluid. The activity retention of the lipase after immobilization was 65% on hydrolysis of p-nitrophenyl butyrate (p-NPB) and the microparticle biocatalyst were stable for at least 6 cycles. Manan et al.102 studied the immobilization of Rhizomucor miehei lipase (RML) on a hybrid support chitosan-chitin with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDAC) as crosslinking agent. The system was used in esterification reaction of eugenyl benzoate and was able to reach higher conversion (56.3% in 5 h) than free RML (47.3%).

2.2.4. Entrapment Entrapment is another technique of irreversible enzyme immobilization as long as the employed support is really insoluble in reaction media. This technique consists of physical confinement of


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enzyme inside the support matrix.21 Usually, carrier and enzyme have not any chemical interaction, unlike the other techniques already discussed, which can avoid any negatives effects associated with the structure of the enzyme.55 It is important to keep in mind that the support must act as a cage ensuring the passage of substrate and product but retaining the enzyme, therefore, support morphology and porosity are essential to the proper functioning of this system.28 In this technique, the support is not prefabricated. It is formed in the presence of the enzyme which becomes entrapped inside it.99 Thus, the support formation conditions need to be compatible with the employed enzyme stability in order to avoid the early denaturation of the biocatalyst.34 Nevertheless, the main disadvantage of this technique may be related with the mass transfer phenomena through the formed support. The diffusion rate of the substrate and the product is often the limiting parameter, and generally, high substrate concentrations are required to minimize its influence.21 Rehman et al.103 immobilized lipase from Penicillium notatum (PNL) by entrapment in silicone polymeric films with efficiency higher than 92% and observed retention about 90% of its original activity after ten cycles of octyl octanoate synthesis reaction. Sodium alginate beads was used by Padilha et al.104 to immobilize lipase from Burkholderia cepacia also by entrapment technique. The immobilized catalyst provided higher conversion yield (92% in 24 h) in the esterification of acetic acid and isoamyl alcohol to produce isoamyl acetate (banana flavor).

2.3. Methods for immobilized lipase characterization As discussed, all techniques for enzyme immobilization may lead to some changes on conformation or surface of enzymes due to the degree of attachment or some else interaction support/enzyme or enzyme/enzyme. Thus, it is almost mandatory to know how the structure of the


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enzyme presents itself following immobilization process in order to evaluate the efficacy of an immobilization technique and development of future strategies. Characterization technologies have been developed following the demand imposed mostly by the advances on new supports, including varied morphologies and nanoscale level, which require obtaining even more accurate information and in brief timing. Among the techniques that have been employed, scanning microscopies are becoming increasingly widespread. It is the case of electron microscopy techniques - field emission scanning electron microscopy (FESEM)/scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and atomic force microscopy (AFM).21 The first group allows the characterization of morphological surface and today is the only technique available to get structural information on materials at nanometer scale resolution. The second one, on the other hand, provides both morphological and mechanical information at the nanometer level, besides allowing to visualize biomolecules.105 Manan et al.102 applied the FESEM and AFM techniques to visualize the surface of the hybrid chitosan-chitin nanowhiskers beads (CS/CNWs) used to immobilize lipase from Rhizomucor miehei (RML). The micrographs from FESEM exposed irregular-shaped white globules that dotted the surface of RML-CS/CNWs and the three-dimensional renditions of the micrographs from ATM showed spherical protuberant spots that spanned all over the film following the immobilization process, revealing clearly the immobilization of RML on the surface of CS/CNWs. Other two techniques quite robust and precise that can be used for immobilized lipase characterization are X-ray photoelectron spectroscopy (XPS) and atomic emission spectroscopy with inductively coupled plasma (ICP-AES). Both techniques allow an elemental analysis of the samples and can be used to detect even traces of enzymes.21 ICP analysis is precisely enough to


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deal with the problem around absolute protein quantification in specific applications.106 Li et al.90 used XPS analysis to sustain the discussion about the immobilization of Burkholderia cepacia lipase on magnetic nanoparticles. The authors verified the presence of element S on the surface of the carrier, which was attributed to cysteine in the lipase. The ICP technique was compared with the classical Bradford method using BSA for the quantification of protein in an interesting study developed by Nicolás et al.106 using Candida antarctica lipase B immobilized in magnetite nanoparticles. The authors found variation up to ten times between the two approaches. Some other interesting analyses that merit highlighting are circular dichroism (CD) spectroscopy,65,107 for structural (secondary and tertiary) and conformational information with reference to lipase-matrix interactions and lipase-lipase interactions, solid-state nuclear magnetic resonance (SSNMR)108 and the classic Fourier transform infra-red spectroscopy (FTIR),11,109,110 to confirm the presence of enzyme attachment on the surface of matrix through assessment of the nature

of

the

chemical

bonds,

and

thermal

gravimetric

analysis

(TGA)37,63

and

microcalorimetry,111 for thermal stability of the immobilized lipase, kinetics of decomposition and catalytic efficiency of immobilized lipase. Thus, as observed, different techniques are extensively used to determine the enzyme structure as well as to confirm presence of enzyme attachment on the surface of different supports during the immobilization process. It is also worth mentioning that new rising methodologies as SSNMR and circular dichroism can be employed for a successful characterization of immobilized enzymes.

3. Immobilized lipases: reactions and products Lipases have been originally designed to catalyze ester bonds via hydrolysis reactions with simultaneous consumption of water molecules. However, considering the principle of reversibility,


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the reverse reaction of ester synthesis also happens on the molecular level.2 The equilibrium between both reactions depends on the water content of the reaction mixture and the combination of these two basic process in a sequential mode can lead to other reactions usually namely transesterification.6 According to substrates available on the reaction mixture it may be termed acidolysis (ester + carboxylic acid), alcoholysis (ester + alcohol) and interesterification (ester + ester).112 Besides, lipases are able to catalyze other reactions with consumption of specific substrates, such as aminolysis and glycerolysis.10 In fact, in all such processes water is needed both for maintenance of the enzyme structural integrity and generation of the catalytic intermediate, being sequentially consumed and formed in the course of the reaction.1 The reactions that can be catalyzed by lipases are schematically represented in Fig. 4. Each reaction represented in this figure is explored below on specific topics with emphasis on the reactional mechanism, application, and products that can be obtained. Independent of the reaction type, the most accepted description of the catalytic action of lipases is a Ping-Pong Bi-Bi mechanism.1 This mechanism means two substrates generate two products (bi-bi) in a no-ordered (ping pong) way, i.e. the lipase releases the first product before to binding of all substrates (see Fig. 5). Two main steps are known and describe the catalytic action of lipases: the first step consists in a nucleophilic attack of the serine hydroxyl group on the substrate ester bond resulting in the formation of an acyl-enzyme and release of the alcohol moiety of the original substrate; the second step is the hydrolysis of the acylated enzyme complex resulting in the formation of the product and the regeneration of the enzyme.1,6 Table 3 summarizes some recent reports related to immobilized lipase applications.


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3.1. Hydrolysis The natural substrates of lipases are triacylglycerols (TAG) of long-chain fatty acids (fats and oils) which via hydrolysis reaction release diacylglycerols (DAG), monoacylglycerols (MAG) and, finally, glycerol, with fatty acids being released at each step (Fig. 6.a).2 These fatty acids may be released either randomly from any position or preferentially from a specific position, depending on the origin of the lipase employed.9 Lipases from Chromobacterium oiscosum, Pseudomonas fluorescens, Candida cylindracea, Geotrichum candidum, and Penicillium eyelopium are considered as nonspecific and release fatty acid in any position. Rhizopus arrhizus lipase, Aspergillus niger lipase, Rhizopus delemar lipase, and Mucor miehei lipase are known as sn-1,3 type since preferentially release the fatty acids from the terminal positions of the glycerol backbone. Meanwhile, sn-2 specificity are extremely rare and refers to preferential release from the center of the structure, such as lipase from Geotrichum candidum.6 As mentioned, the hydrolysis is a reversible reaction and the chemical equilibrium may be displaced to the production of fatty acids and glycerol using higher water content on the system.1 The fatty acids are the most important product of this reaction and can be used for the manufacturing of soap, cosmetics, and food emulsifying agents. Recently, the interest by polyunsaturated fatty acids (PUFA), especially long-chain omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is increasing due to their considerable health-promoting effect.15 They are used as ingredients in dietary supplements, healthy foods, and pharmaceutical products in the prevention and treatment of cancer, arteriosclerosis, inflammation, and hyperlipemia, for example.113,114 Urrutia et al.51 produced longchain omega-3 polyunsaturated fatty acids (n-3 PUFA) from hydrolysis of menhaden oil by lipase B from Candida antarctica and lipase from Rhizomucor miehei immobilized in chitosan particles.


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Glycerol is considered a valuable by-product of the hydrolysis reaction with a wide range of industrial applications. Glycerol has over two thousand different applications and can be sold mostly as a commodity to pharmaceuticals, personal care, food, and cosmetics industries where can be used to dissolve drugs, as carrier for antibiotics and antiseptics, as plasticizer or lubricant, besides being the major ingredient in toothpaste.115 A review published by Tan et al.115 reported the applications of glycerol, as well as the various production routes, including hydrolysis reaction. Nowadays, immobilized lipases have been used in the so-called added-value fine chemistry applications, mostly in the pharmaceutical field, e.g., on the resolution of chiral compounds116 Chiral non-racemic compounds are important substrates for the synthesis of numerous drugs, cosmetics and model compounds. The use of lipases is mainly due to the characteristics of the regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors.

117–119

However, obtaining a pure enantiomer at the end of the synthesis is still a

challenge task.120 Recently, Borys et al.121 successfully synthesized 2-benzyl-3-butenoic acid 3 (98% yield) using the commercial immobilized lipase Novozym 435 (Candida antarctica lipase B). The authors designed a protocol sequentially combining the esterification and hydrolysis reactions taking into account the selectivity of the lipase.

3.2. Esterification The esterification reaction is the reverse reaction of the hydrolysis and occurs spontaneously and simultaneously. In the case of TAG hydrolysis generating fatty acids and glycerol, the reverse reaction is the synthesis of MAG, DAG, and even TAG coming from fatty acids and glycerol releasing water. This direction of reaction can be favored by low water content on the system, and the degree of esterification can be controlled by removal of free water.2 Currently, MAG are the


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most used emulsifiers in food, cosmetic and pharmaceutical industries,122 whereas DAG have shown a good capacity of preventing obesity and of other lifestyle-related diseases.123 Although MAG and DAG could be obtained via esterification reaction, their production is almost totally via glycerolysis reaction, thus, more emphasis on these compounds is given on a specific section. Itabaiana et al.124 studied the synthesis of MAGs, specifically monostearin, from the esterification reaction of the stearic acid with glycerol catalyzed by Candida antarctica lipase B immobilized in AOT/isooctane and high conversion (80% in 30 min) was achieved. Although the esterification reaction is originally related with the reversibility of the hydrolysis reaction of TAG, in 1979 this reaction (by enzymatic route) started to be investigated as a possibility of getting other derived products, in that case organic esters, used in many industrial applications.125 Since then, esterification reaction has been investigated and has drawn growing increasing attention as a reaction between an alcohol and a carboxylic acid generating an ester and water.10 Flavors and fragrance esters for food applications are an important consumer market of the esterification reaction because of the difficulties and high costs of extracting flavor from their natural sources. The same scene is seen in cosmetic industries where perfume esters, such as 2phenethyl acetate, ethyl caproate, and isoamyl acetate, are demanded.116,126 Wax esters for personal care products, candles, board sizing, lubricant, coatings, packaging, and food can be obtained by using long-chain carboxylic acids and long-chain alcohols.127 The main question, in this case, is the water which must be almost totally removed from the system to allow high conversion.15 Yadav and Kamble86 synthesized numerous esters of geraniol, including geranyl acetate, catalyzed by various commercial immobilized lipases, namely, Novozym 435 from Candida antarctica, Lipozyme TL IM from Thermomyces lanuginosus and Lipozyme RM IM from Rhizomucor


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miehei. The high conversion, 96%, was reached when Novozym 435 was used. Papadaki et al.127 reported the employ of microbial oils produced by the oleaginous yeasts Cryptococcus curvatus, Rhodosporidium toruloides and Lipomyces starkeyi, as feedstock for the production of wax esters catalyzed by commercial immobilized lipases Novozym 435 from Candida antarctica and Lipozyme from Mucor miehei using cetyl, oleyl and behenyl alcohols. The production of biodiesel by enzyme-catalyzed process has received increasing attention because it requires lower energetic demand than the conventional alkali-catalyzed technique and the substrates can be processed without any pretreatment.10,128 Currently, China and Brazil are leading the industrial applications of lipase-catalyzed approach toward biofuels production.129 The enzymatic production of biodiesel by esterification of fatty acids using immobilized lipases was reported by several authors.11,130,131 Water is the main by-product of esterification and, again, needs to be removed or, alternatively, large amounts of alcohol could be used to shift the reaction to ester formation.10 Although more commonly obtained by hydrolysis reaction, enantiomers to pharmaceutical industries can equally be synthesized by esterification reaction. Recently, Brodzka et al.120 developed a study on the enzymatic kinetic resolution of 3-phenyl-4-pentenoic acid with methanol and various trialkyl orthoesters employing the commercial immobilized lipase Novozym 435 (Candida antarctica lipase B). Unfortunately, the use of methanol afforded very poor results and only traces of racemic product were obtained. The authors attributed this to the inactivation of enzyme in the presence of short-chain alcohols. On the other hand, Verri et al.132 used the same route to chiral resolution of (R,S)-ibuprofen employing the Rhizomucor miehei lipase immobilized onto hybrid silica nanospheres and 1-propanol as alcohol, achieving ester yield ranging between 78 and 93%. According to the authors, this yield is quite interesting taking into account the fact


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that only the (S) enantiomer of ibuprofen was synthesized, thus demonstrating the high enantioselectivity of the lipase applied.

3.3. Transesterification Transesterification is the general term used to name three reactions derived from the sequential combination of the hydrolysis and esterification reactions. It can be acidolysis (when an acyl group is displaced between an ester moiety and a carboxylic acid moiety), alcoholysis (when an acyl group is displaced between an ester moiety and an alcohol moiety) and interesterification (when two acyl groups are exchanged between two ester moieties). This concept of transesterification causes divergence between researchers since decades ago and even presently it still without a total agreement as some researchers call the reaction ester + ester of transesterification and the general term interesterification. A way of understanding this concept is to assume all the reactions as transfer or exchange of acyl moieties, so the transesterification as the general term may sound better.1,2,112

3.3.1. Acidolysis Acidolysis involves an acyl transfer between an ester and carboxylic acid, i.e. a triglyceride and fatty acid. In other words, acidolysis allows incorporation of specific fatty acids at specific positions on the triglyceride molecule. This reaction is important in oil modification and preparing functional lipids termed structured lipids (SLs).116 Structured lipids are TAGs that have been restructured to change the positions and composition of fatty acids from the native state.133 They have been developed to meet the demand of health-conscious consumers as can be tailor-made to target specific diseases and metabolic conditions.134–136


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Chojnacka and Gładkowski137 studied the production of Structured Phosphatidylcholine by means the acidolysis reaction between phospholipids from egg yolk and myristic acid using four commercial immobilized lipases as catalyst. At the same time, the authors performed interesterification reactions in order to evaluate the better production route. Abed et al.138 investigated lipase-catalyzed acidolysis of microbial oil from Mortierella alpina with caprylic acid to produce structured lipids enriched with medium-chain fatty acids. These structured lipids are readily absorbed via the portal vein and oxidized for energy, and generally are less likely to be accumulated in the body as stored fat. In this work, four commercial immobilized lipases were also screened for their acidolysis efficiency and Lipozyme RM IM from Rhizomucor miehei displayed the highest ratio of CA incorporation. Li et al.135 investigated the improvement of acidolysis activity of the Rhizopus oryzae lipase immobilized in NKA-9 resin.

3.3.2. Alcoholysis Alcoholysis reaction concerns an acyl transfer between an ester and alcohol.2 Among transesterification reactions, alcoholysis is the most widespread and researched one. In the last ten years, it is estimated more than half of publications involving these terms were destined for biodiesel production, especially synthesis of methyl and ethyl esters. Because of this, it is quite common to found works using only the generic term transesterification to label this reaction. On the alcoholysis, a triglyceride and a short-chain alcohol are used forming alkyl esters of fatty acids and glycerol for example (see Fig. 6.b). Remonatto et al.139 investigated the performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different hydrophobic supports. The biocatalysts obtained were employed in the production of FAME and FAEE (fatty acid ethyl ester) via alcoholysis of sunflower oil exhibiting good reaction


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conversions. Recently, some researchers have investigated the operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction.140,141 It was observed that the Novozym 435 chemically modified by 2,4,6- trinitrobenzensulfonic acid (TNBS), ethylendiamine (EDA), or polyethylenimine (PEI) became more resistant to rupture besides be fully active in alcoholysis reaction.140 The broken particles of biocatalyst may cause problems of blockage in filtration operations and column reactors. Some researchers have dedicated their efforts to clarify the mechanism of alkyl esters synthesis under two points of view. In the first, lipase synthesizes esters by direct alcoholysis of TAGs in a single step. The second one involves the hydrolysis of TAGs and subsequent esterification of the resulting fatty acids.9,128,142,143 Apparently, a route is preferred, but not exclusive, to another according to the lipase employed and its specificity, i.e., the hypothesis of this transesterification is a combination of those two viewpoints occurring simultaneously seems appropriate.128 If we think in alcoholysis reaction, at least partly, as the same as hydrolysis followed by esterification, it is possible to realize that almost all the same final products of esterification reaction can be obtained by lipase-catalyzed alcoholysis. It is the case of MAGs and DAGs and flavor compound both already discussed in the esterification section but which still deserving attention. Muñío et al.144 synthesized 2-monoacylglycerols (2-MAG), rich in polyunsaturated fatty acids (PUFAs) by alcoholysis of fish oils with ethanol, catalyzed by lipase Rd from R. delemar and lipase D from Rhizopus oryzae both immobilized on Accurel MP 1000, besides the commercial immobilized lipase Novozym 435 from Candida antarctica and they achieved 2-MAG yields higher to the first two immobilized (>70%) than that obtained with the commercial immobilized (

Driving immobilized lipases as biocatalysts: 10 years state of the art

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